We propose to continue our studies of the molecular mechanisms involved in the replication of bacteriophage Gamma DNA and its regulation. Our long range goal is to elucidate the biochemical roles of each of the numerous Gamma and Escherichia coli replication proteins which participate in this complex, multienzyme process. A major focus of our research will be an investigation of the properties of the replication of supercoiled Gamma dv plasmid DNA in a soluble in vitro system containing purified Gamma O and P replication proteins. Using electron microscopic analysis techniques, we will locate the site(s) on the Gamma dv chromosome where replication is initiated, determine if the replication is unidirectional or bidirectional, and identify the nature of the Gamma dv replication intermediates. We will use the in vitro Gamma dv replication system to develop assays that will enable us to purify and characterize previously unidentified bacterial replication proteins that participate in Gamma DNA replication. We will constract Gamma dv plasmids, containing promoters located near the Gamma replication origin, for use in studies of the nature of the transcription requirement for "activation" of the Gamma origin. An investigation of the molecular mechanism of the Gamma O and P protein-dependent replication of phage M13 single-stranded DNA will be pursued. We will determine the physical and functional properties of the purified Gamma O protein and characterize its interaction with wild type and mutant Gamma replication origins. We will attempt to separate the O protein into its predicted amino and carboxyl terminal domains. We will initiate studies designed to document functional interactions between the Gamma and O and P replication proteins. Ultimately, we intend to reconstitute a Gamma dv replication system solely with purified proteins and then begin a detailed investigation of the molecular events which occur in this process. This bacteriophage Gamma replication system is an excellent mechanistic model system for studies of the initiation of eukaryotic DNA replication at unique chromosomal origins and for probing the nature of host-virus interactions.